The major neutral, glycolipids and phospholipids from envelopes of spinach chloroplasts were analyzed with respect to proportions, positional distribution and pairing of fatty acids. All specificities in the diacylglycerol portions of lipids known from previous analyses of lipids from whole leaves were also found in envelope lipids. Diacylglycerols and galactolipids share a common diacylglycerol portion. The only exception is digalactosyl diacylglycerol, which contains 18 : 3/16 : 0 but lacks 18 : 3/16 : 3 species reverting the distribution in other galactolipids. Phosphatidylcholine, phosphatidylglycerol and sulfoquinovosyl diacylglycerol are distinct from the galactolipids, because each one has a unique diacylglycerol profile. The diacylglycerol species present in phosphatidylcholine and galactolipids or free diacylglycerols do not provide evidence for a biogenetic relation between phosphatidylcholine and galactolipids at the level of envelopes.

Young leaves from three plants which accumulate hexadecatrienoic acid rather specifically in the sn-2-position of monogalactosyl diglyceride (MGD) were incubated with radioactive CO2 or acetate to investigate the origin of this specificity. Labelled glycerolipids were extracted and analyzed for time-dependent changes of radioactivity in their fatty acids. The investigation of labelled MGD and digalactosyl diglyceride (DGD) included determination of radioactivity in hydrolysis products, separation of molecular species by argentation chromatography and analysis of the positional distribution of fatty acids. The results agree with previous observations on the accumulation of radioactive oleic acid in phosphatidyl choline (PC) and formally with the possibility of a PC-coupled desaturation to linoleic acid. They do not support the proposed function of PC as donator of polyenoic acids. Instead the radioactivity patterns may be interpreted as pointing to a relation between fatty acid desaturation and many if not all glycerolipids, although a different interpretation is also possible. Fatty acid patterns in lipids and their labelling indicate the existence of several pools for 1) MGD, from which only that without C16-unsaturated fatty acids is accessible for galactosylation to DGD ; 2) palmitic acid, from which one part is accessible to desaturation via C16:0 and C16:2 to C16:3 . Since these acids are found labelled in the sn-2-position of MGD, the specific positioning may be related to this separation of C16:0pools. Desaturation of C16:0 seems to be the major source of C16:3; 3) linolenic acid, from which those parts present in the sn-2-position of galactolipids or in PC are characterized by a strikingly slow labelling.

Spinach chloroplasts were purified on gradients of Percoll which preserved envelope impermeability and CO2-dependent oxygen evolution in the light. Application of (35)SO4″ to purified chloroplasts resulted in a light-dependent labeling of a lipid component which was indentified as sulfoquinovosyl diacylglycerol. Fractionation of chloroplasts showed that after 5 min of labeling most of the newly synthesized sulfolipid was present in thylakoids. Only a small percentage was recovered from the envelopes. Molecular species from envelopes and thylakoids were identical. The molecular species did not change during incubation times ranging from 5 min up to 4.5 h. Mesophyll protoplasts from (35)SO4″-labeled oat primary leaves were gently disrupted and separated into organelles by sucrose gradient centrifugation. Labeled sulfolipid was located almost exclusively in the chloroplasts. This, in combination with the experiments carried out with isolated chloroplasts, indicates that the final assembly steps in the biosynthesis of sulfolipid are confined to the chloroplasts.

Lipid mixtures from chloroplast envelope and thylakoid membranes were isolated after different labelling times in vivo and in vitro and separated into major components. The isolated compounds were subjected to analyses such as separation of molecular species, determination of radioactivity in fatty acids and water-soluble hydrolysis products and radio gas-liquid chromatography of fatty acid mixtures. In the case of monogalactosyl and digalactosyl diacylglycerol these procedures were also applied to several individual molecular species. To investigate the extent of de novo synthesis these species were also used for methylation studies and their fatty acids subjected to a-oxidation. In envelope membranes diacylglycerols and monogalactosyl diacylglycerols may each be separated into several distinct and non-mixing pools. Molecules made de novo with oligoene fatty acids are very efficient substrates for galactosylation in vivo. The time-dependent changes in patterns of galactolipid molecular species may indicate a desaturation of acyl chains operating in close contact to intact lipids. After isolation, envelopes incorporated UDP-['4C]galactose into completely different patterns of galactolipids and molecular species pointing to changed properties of this membrane system or to a loss of regulatory factors.In the preceding paper [I] we presented data on the distribution of radioactive lipids between envelopes and thylakoids after labelling of chloroplasts in vivo. Apart from our interest in intracellular lipid exchange processes we continue trying to find out how and where specificities of fatty acid patterns are introduced into chloroplast lipids. In previous experiments we had approached this problem by analyzing labelled lipids extracted from whole leaves [2]. More conclusive results were expected from an analysis at the level of organelles and membranes. Therefore we carried out a detailed analysis of unlabelled envelope lipids with respect to positional distribution and pairing of fatty acids [3]. All specificities established before by analyzing extracts from whole leaves [4] were also found at the level of envelopes. In particular, the difference in the diacylglycerol moieties of monogalactosyl and digalactosyl diacylglycerol was also observed in envelope membranes, which are supposed to be the main intracellular site of galactolipid biosynthesis [5]. This difference is surprising in view of the simple galactosylation reaction linking both compounds. In the present communication we report details of the timedependent labelling patterns of lipids from envelopes and thylakoids labelled in vivo and in vitro since we expected to observe the gradual appearance of the above-mentioned specificities especially in envelope membranes. MATERIALS AND METHODS General Methods and Lipids from EnvelopesLabelled in vivo. Lipid extracts were prepared in the same way from the same samples as described in the preceding publication [l].IFlCUbUtionS with Envelopes in vitro. Envelopes were isolated from field-grown spinach according ...

Spinach leaves were labelled with I4CO2 for subsequent isolation of radioactive chloroplasts, which were separated into envelopes and thylakoids. The analyses carried out with the lipid extracts from these membranes were concerned with the following questions : are chloroplast envelopes a functional interface between endoplasmic reticulum and thylakoids, do they also play a predominant role in galactolipid biosynthesis in vivo, and is it possible to demonstrate galactolipid export from envelopes into thylakoids?Taken together the results show that lipid export is apparently too fast in vivo to be followed by the labelling and separation technique used, since thylakoid lipids contained always far more total label than envelope counterparts, whereas the specific activity of envelope lipids was higher. Phosphatidylcholine, which has been suggested to function as acyl carrier between endoplasmic reticulum and chloroplasts, was never found labelled to any extraordinary extent in envelopes. Envelopes may be regarded as small, but rapidly turned over lipid pools.